Optimized protocol for the generation of an orthotopic colon cancer mouse model and metastasis

Summary The microenvironment plays an essential role in tumor development and metastatic progression. Here, we describe a simple and rapid protocol to generate tumors in mice using colon cancer cell lines or tumoroids in the correct microenvironment, colonic mucosa. We also detail steps for monitoring the growth of the primary tumor in real time using colonoscopy or in vivo imaging system, as well as monitoring metastasis development. Finally, we describe tissue collection and sample preparation for subsequent immunohistochemistry analysis.


SUMMARY
The microenvironment plays an essential role in tumor development and metastatic progression. Here, we describe a simple and rapid protocol to generate tumors in mice using colon cancer cell lines or tumoroids in the correct microenvironment, colonic mucosa. We also detail steps for monitoring the growth of the primary tumor in real time using colonoscopy or in vivo imaging system, as well as monitoring metastasis development. Finally, we describe tissue collection and sample preparation for subsequent immunohistochemistry analysis.

BEFORE YOU BEGIN
The tumor microenvironment plays critical roles during tumor development, cell dissemination and metastasis formation. The development of colon cancer in the correct microenvironment could be studied using transgenic animals or carcinogen/inflammation-induced tumor mouse models. However, these models are slow and rarely recapitulate tumor progression until metastasis. Alternative models include injecting colon cancer cells subcutaneously or in the cecum; however, that does not recapitulate the natural microenvironment for colon cancer cells. While several publications have recently described the transplantation of normal organoids into injured colonic mucosa, 1,2 here we describe a protocol that enables the generation of primary colonic tumors that can metastasize into the liver. This rapid and reproducible protocol also significantly reduces mouse mortality due to the procedure.

Institutional permissions
This protocol requires the use of mice. All mouse experiments should be performed following relevant governmental and institutional guidelines. Animals used in this study were maintained in the SPF animal facility of Institut Curie (Paris, France) before use. All experiments described in this study were performed in accordance with the European and French National Regulation for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (2010/63/UE) for the care and use of laboratory animals. All experimental procedures were approved by the ethics committee of the Institut Curie CEEA-IC #118 amendment to the projects 2020-002 (Authorization: #25603-2020053122444776-v2) and 2020-010 (Authorization: #27460-2020100614277480-v1) given by the National Authority in compliance with the international guidelines.

Expansion of cell lines and organoids
Timing: 2-7 days (for step 1) Timing: 3-7 days (for step 2) The protocol described in this paper is based on commercial human colon cancer cell line SW480 and ''in-house'' mouse tumor organoids (tumoroids). We have also used this protocol successfully with other colorectal cancer cell lines, such as mouse cell lines CT26 and MC38 and human cell lines HCT116, HT29, and SW837, as well as mouse intestinal organoids from tumor tissue.
a. Culture the cells in DMEM Glutamax medium supplemented with 10% Fetal calf serum in 75 cm 2 culture flasks until 80% confluence. b. Split cells 1:5 twice a week.
Note: This is sufficient to inject about 20 mice (0.5 million cells per mouse).
Note: Before starting the experiment, expand tumoroids to obtain 12 wells of 24-well plate to have 0,5 million cells per mouse.
a. Detach Matrigel drops by scratching using the tip of a 1 mL pipette. b. Aspirate the drop and the medium using a 1 mL pipette. c. Transfer to a 15 mL tube and add medium until 10 mL to balance the centrifuge. d. Spin down cells for 10 min at 450 g at room temperature. e. Aspirate the supernatant. f. Add 1 mL of Tryplee Express (room temperature) to the pellet. g. Incubate for 3 min at 37 C, 5% CO 2 , until tumoroids dissociation. h. Add 5 mL medium with 10% FBS to inactivate Tryplee Express. i. Spin down cells for 10 min at 450 g at room temperature. j. Aspirate the supernatant. k. Resuspend cell pellets in 50:50 Matrigel/tumoroids culture medium mix on ice to prevent premature Matrigel polymerization. l. Deposit 50 mL drops at the bottom of the 24-well plate pre-wormed at 37 C to speed up Matrigel polymerization and thus prevent flattening of the drop. m. Put the plate upside-down at 37 C, 5% CO 2, for 30 min to polymerize Matrigel.

Timing: 7 days
Note: We graft mouse tumoroids and SW480GFP-LUC cells in Nude NMRI mice. However, for other cell lines, we used mice with different backgrounds to match the background of each cell line. For example, graft MC38 cells in C57BL/6 mice, and CT26 cells in Balb/c mice.
Note: We use six weeks old mice, both male and female. Acclimatize mice in the animal facility for at least one week before injection.

Timing: 7 days
Note: The culture media for the tumoroids should be used fresh and thus prepared on the same day when tumoroids are amplified and prepared for injection. The EDTA solution should be placed in a water bath heated to 50 C for 15 min before the injection of the cells.

MATERIALS AND EQUIPMENT
Recipes are provided to a specified final volume, but investigators may choose to prepare different volumes depending on the number of samples processed.

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Note: The dose injected per mouse is 5 mg/kg.

STEP-BY-STEP METHOD DETAILS Preparation of cells for injection (day 0)
Timing: 10-30 min Timing: 10 min (for step 1) Timing: 30 min (for step 2) This section describes the preparation of cells that will be injected into the mouse colon.
Note: We injected 0.5 million cells (SW480 or tumoroids) per mouse. i. Take 10 mL of cell suspension and add 10 mL of trypan blue.

Solution of Carprofen
ii. Deposit this mix on cell counting chamber slides and count cells with cell counter.
iii. Calculate the volume of cells needed to obtain 0.5 3 10 6 cells in 100 mL that will be injected in each mouse.
Note: Routinely, cell viability is above 95%. If cell viability is less than 80%, do not use these cells. Note: In our experience, 2 h of waiting did not affect tumor engraftment.

Prepare cells from tumoroids.
a. Detach Matrigel drops by scratching using the tip of a 1 mL pipette. b. Aspirate the drop and medium using a 1 mL pipette. c. Transfer all drops to a 15 mL tube. Then add medium until 10 mL to balance the centrifuge. d. Spin down cells for 10 min at 450 g at room temperature.
Note: room temperature is in range of 19 C-22 C.
e. Aspirate the supernatant. f. Add 1 mL of room temperature Tryplee Express to the pellet. g. Incubate for 3 min at 37 C, 5% CO 2 , then assess under the cell culture microscope that tumoroids are dissociated into single cells or small clusters. h. Add 5 mL medium to inactivate Tryplee Express. i. Count cells: i. Take 10 mL of cell suspension and add 10 mL of trypan blue.
ii. Deposit this mix on cell counting chamber slides and count cells with cell counter.
iii. Calculate the volume of cells needed to obtain 0.5 3 10 6 cells in 100 mL that will be injected in each mouse.
Note: If there are small clusters, count the cells in each cluster. If cell viability is less than 80%, do not use these tumoroids.
j. Spin down cells for 10 min at 450 g at room temperature. k. Discard the supernatant and resuspend cells in 100 mL for each mouse of a solution made of 50:50 culture medium (DMEM/F12 without FBS) and Matrigel on ice to prevent premature polymerization of Matrigel. l. Keep cells on ice until injection into mice. In our experience, 2 h of waiting did not affect tumor engraftment.

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Preparation of mouse (day 0)

Timing: 5 min per mouse
This section describes the preparation of mice before cell injection. The mouse needs to be immobilized and anesthetized. An analgesic is given to mice before the procedure to reduce pain. The colon should be cleaned to remove faces, which is done by gently massaging the mouse's abdomen.
3. Anesthetize the mouse with 3% isoflurane and 2 L per minute air flow until it falls asleep. 4. Once the mouse is sleeping, reduce isoflurane to 2% with an airflow of 0.5 L per minute. 5. Place tear gel on the eyes of the mice to prevent drying. 6. Inject 5 mg/kg Carprofen (Rimadyl) analgesic (in 100 mL) diluted in PBS subcutaneously. 7. Position the mouse on its back with hind legs apart, immobilized with surgical tape ( Figure 1A). 8. Remove feces present in the colon by gently massaging the mouse's abdomen.
Note: You should feel with fingers that the colon is empty.
CRITICAL: This step is essential because unremoved feces in the distal part of the colon may prevent the cells from attaching to the colonic mucosa. Feces can also open the anus too quickly (which will be obstructed at the end of the experiment using surgical glue) and thus decrease the time of adhesion of cells to the colonic mucosa. Do not wash the colon with PBS or similar solutions, as this will dilute the EDTA and the injected cells.
Removal of mouse epithelium (day 0) Timing: 5 min This step injures the colonic mucosa to felicitate the attachment of injected cells. The lesions are made by an electric interdental toothbrush soaked into a hot EDTA solution. The same experimenter can prepare both the mice and EDTA.
9. Pre-heat 0.5 M EDTA, pH8 at 50 C in a water bath 15 min before the start of the experiment. 10. Immerse the interdental brush for 30 s in the pre-heated EDTA solution ( Figure 1B). 11. Gently insert the interdental brush into the mouse's anus and advance it 1.5 cm into the colon ( Figure 1C). 12. Turn on the vibration of the interdental brush for 10 s.
CRITICAL: This step is crucial because the number of removed crypts must be sufficient but not excessive. The optimal number of detached crypts is between 2,500 and 5,000 crypts. The pressure exerted on the interdental brush should not be too strong to avoid piercing the colonic wall. In case of bleeding, interrupt the abrasions immediately.
13. The interdental brush is gently removed and immersed in an Eppendorf tube containing 1.5 mL of PBS ( Figure 1D). 14. Turn on the vibration for 5 s to release the removed crypts ( Figures 1E and 1F

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Live imaging of tumor growth by endoscopy (day 7 to day 90)

Timing: 5 min per mouse
This step allows visualization of tumor growth by colonoscopy ( Figure 3). Colonoscopy could be used to observe a healthy, non-damaged colon ( Figure 3B), a colon just after damage with an interdental brush ( Figure 3C), or to follow tumor growth ( Figures 3D and 3E).
Imaging is done starting one week after the transplantation, once every week and up to 3 months, depending on the tumor growth. Mice could be kept alive until the defined humane endpoint, i.e., 50% obstruction of the colonic lumen or deterioration in the general condition of the animal.
Note: Repeating the imaging several times a week should be avoided to limit repeated anesthesia and limit the risk of bleeding in the animals.
27. Anesthetize mice using 3% isoflurane, 2 L per minute, until it falls asleep. 28. Reduce isoflurane between 2% isoflurane with airflow of 0.5 L per minute during imaging. 29. Immobilize the mouse with tape on the hind legs. 30. Remove feces by abdominal massage as described above.
Note: You should feel with fingers that the colon is empty.
31. Apply vaseline around the anus to facilitate insertion of the endoscope. 32. Insert the endoscope gently into the anus while dilating the colon by blowing air into the port using a 50 mL syringe ( Figure 3A). The quantity of air depends on how close the colon is. In general, the injected volume of air is around 5 mL. 33. Move the endoscope throughout the colon gently while recording the movie.

Live imaging of tumor growth intravital IVIS (day 7 to day 90)
Timing: 30 min per mouse This step allows visualization of tumor growth by IVIS (Figure 4). IVIS could be used to follow the growth of the primary tumor by repeated imaging (Figures 4C-4E) or to visualize metastasis (Figure 4F). Imaging is done starting one week after the transplantation, once every week and up to 3 months, depending on the tumor growth.    Note: The OCT block should be trimmed until reaching the tissue.
54. Trim the anus and rectum until reaching the tumor that can be seen macroscopically. 55. Cut colon/tumor into 8 to 10 mm-thick slices.
Note: For other tissues, just trim the OCT and proceed with cutting the tissue into 8 to 10 mmthick slices.
56. Transfer the tissue to the slide Super frost + by contact.

Staining samples (day 30 to day 90)
Timing: 2 h 57. Add 500 mL of a solution containing 25 mg/mL DAPI in PBS to the sections. 58. Incubate for 30 min at room temperature. 59. Remove the DAPI solution, add 8 drops of Aquapolymount mounting solution on the tissue section and cover with 24 3 50 mm coverslip. 60. Leave to dry for at least 1 h before imaging.

Imaging samples (day 30 to day 90)
Timing: 1 h per sample 61. Image samples using an epifluorescent microscope or, for higher resolution images, a confocal microscope ( Figures 5B and 6C-6F).

Analysis of the primary tumor and metastasis
Here we describe the results obtained with the tumoroids injected in Nude mice ( Figure 5) colorectal cancer line SW480GFP-LUC and ( Figure 6). In both cases, primary tumors developed in the distal colon, very close to the rectum ( Figure 6A). During the autopsy, peritoneum and liver metastases were detected only in the mouse injected with SW480 cells ( Figure 6B). To study the morphology of these tumors, 10 mm thick transversal sections of frozen tissue were made using a cryostat and deposited on slides. Nuclei were stained using DAPI, and the expression of GFP identified tumor cells. The entire transversal section of the colon was imaged using a LISPI confocal microscope ( Figure 6C).

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Higher magnification images of the primary tumor and metastasis were taken using an LSM880 confocal microscope ( Figures 6D-6F).

Extension of the method to other cell lines and mouse strains
First, we tested if other mouse strains could be used with this procedure. We found that the Balb/c mouse strain is more sensitive to brush injury than C57BL/6 and Nude mice. While 50% of Balb/c mice (n = 16 mice) died one day after the procedure, the mortality rate was lower for other mice strains -only 21% of C57BL/6 (n = 102 mice) and 12% of Nude (n = 114 mice).
Second, we successfully used the protocol with other human colorectal cancer cell lines, such as HT29, HCT116, and SW837, injected in Nude mice, and mouse colon cancer cells, CT26 and MC38, injected in mice with matching genetic backgrounds: CT26 cells were injected in the Balb/c mouse strain, while the MC38 were injected in C57BL/6. Finally, we tested the protocol with primary human colon cancer cells isolated from patient-derived xenografts (PDX). We generated cell suspension by enzymatically dissociating PDX tumors, purifying live cells, and injecting them into Nude mice. While all models give rise to tumors, the efficiency of tumor development ranges from 25 to 100%.
Third, we tested if there is a relationship between the number of injected cells and the efficiency of tumor development. We injected either 0.5 or 2 million MC38 cells into C57BL/6 mice. We found that the higher number of injected cells leads to a higher percentage of mice that develop tumors. 2 million cells generated tumors in 89% of mice (n = 28), while 0.5 million cells generated tumoris in 31% of mice (n = 27).
Fourth, we assessed if the number of released crypts correlates with the efficiency of tumor development. We found that tumor development does not depend on the number of detached crypts for a high number of injected cells (2 3 10 6 cells). However, for a lower number of injected cells (0.5 3 10 6 cells), a higher percentage of tumors were obtained with 2,500 and 5,000 detached crypts (data for MC38 cells injected in C57BL/6 mice).
Fifth, we assessed the robustness of the protocol. As mentioned above, when injected with 2 million MC38 cancer cells, about 90% of mice developed tumors assessed by colonoscopy and confirmed by histological examination after necropsy. All mice needed to be sacrificed two weeks post-injection as tumors obstructed more than 50% of the colonic lumen. At that time, tumors were approximal the same size. If one wants to evaluate whether a specific gene product affects tumor initiation, that gene could be overexpressed or deleted in cancer cells before the injection. To calculate the number of animals required for each group, one can use the Fleiss formula. 6,7 For example, if tumor incidence decreases to 50% and one would like to have an 80% chance of detecting this decrease, testing at p = 0.05, 14 animals per group are required. The tumor incidence when injecting 250 000 SW480 cells is 83%. Thus, using the same parameters, we calculate that 24 mice per group are required. Metastasis incidence in this model is 50% of mice. If incidence of metastasis increases to 80%, one would need 45 mice per group.

LIMITATIONS
All tested cell types (human and mouse cell lines, dissociated tumoroids and cell suspension from PDX) can give rise to orthotopic tumors using this method. However, we noticed some limitations.
First, mouse genetic background may be a limiting factor. We found that the Balb/c mouse strain is sensitive to brush injury of colonic mucosa. Thus, we recommend the use of Nude and C57BL/6 mouse strains. Other strains must be tested for sensitivity to colonic injury before starting the protocol. Second, metastasis development is inefficient. We observed metastasis only with SW480 cells injected in Nude mice. The lack of metastasis is likely due to the rapid growth of primary tumors. In most models, the primary tumors grow rapidly, reaching the size when the mouse must be sacrificed due to ethical reasons, which is insufficient time to establish metastasis.
Third, as it is hard to control the number of attached cells and as tumoroids coming from different mouse models could have different attachment abilities, it is hard to compare tumor growth using different tumoroids. Thus, tumor initiation, growth and metastasis could be only compared between the same parental cell lines or tumoroids. Imaging mice using IVIS one day after injection is important to ensure that the similar number of cells are grafted.

TROUBLESHOOTING Problem 1
The colon is not cleaned of feces. Feces descend quickly and push the cells outward before they can attach to the wounded mucosa (steps 8 and 30).

Potential solution
Massages of the mouse's abdomen using fingers and pressures on the rectum must be performed to force the feces out of the colon. Wait for 10 min for all feces to exit.
If the colon is too full, which is felt under your fingers, proceed to the next mouse, while this one empties its colon naturally.
Do not wash the colon with PBS, as this dilutes the cell suspension.
Note: We tried to deprive mice of food 12 h before injection. Even though this reduced the number of feces in the colon, the mice were too weak, and the percentage of mortality increased.

Problem 2
The mouse rectum is still blocked after 4 h due to excessive glue (step 26).

Potential solution
Gently remove the glue with a damp cloth, avoiding tearing the tissue.
Limit the amount of glue (around 20 mL-25 mL) when closing the anus.

Problem 3
Sometimes, the growing tissue mass observed during endoscopy could be misinterpreted as a growing tumor. However, during microscopic analysis, this mass was identified as a local inflammation reaction due to the injury, not a tumor (step 33).

Potential solution
Microscopic observation of the growing tissue mass is required to differentiate an inflammatory reaction from the tumor.

Potential solution
High mortality for some mouse strains could be due to a bigger extent of the injury. As it is impossible to change the vibration speed on the electric interdental brush, the only possible solution could be to decrease the number and duration of manual injuries.

Problem 5
The tumor grows too fast, obstructing the colon, and thus mouse needs to be sacrificed before metastases are developed (step 33).

Potential solution
Decrease the number of cells to be injected.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact: danijela.matic@curie.fr. All questions about the technical specifics of performing the protocol should be addressed to technical lead contact: sophie.richon@curie.fr.

Materials availability
Only SW480GFPLUC cells were newly generated for this protocol, and they are available for the community. All other materials mentioned above are commercially available.

Data and code availability
This study did not generate datasets code.